Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis

Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm. Subtraction of consecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling. Tandem scanning confocal microscopy image analysis of living cells in real time provides repetitive spatial, temporal, and quantitative information about cell adhesion. Such an approach should allow more precise quantitative analyses to be made of the interactions between extracellular matrix, adhesion proteins, integrins, and the cytoskeleton in the living cell.